Secreted factors from M1 macrophages drive prostate cancer stem cell plasticity by upregulating NANOG, SOX2, and CD44 through NFκB-signaling.

The inflammatory tumor microenvironment (TME) is a key driver for tumor-promoting processes. Tumor-associated macrophages are one of the main immune cell types in the TME and their increased density is related to poor prognosis in prostate cancer. Here, we investigated the influence of pro-inflammatory (M1) and immunosuppressive (M2) macrophages on prostate cancer lineage plasticity. Our findings reveal that M1 macrophage secreted factors upregulate genes related to stemness while downregulating genes associated with androgen response in prostate cancer cells. The expression of cancer stem cell (CSC) plasticity markers NANOG, KLF4, SOX2, OCT4, and CD44 was stimulated by the secreted factors from M1 macrophages. Moreover, AR and its target gene PSA were observed to be suppressed in LNCaP cells treated with secreted factors from M1 macrophages. Inhibition of NFκB signaling using the IKK16 inhibitor resulted in downregulation of NANOG, SOX2, and CD44 and CSC plasticity. Our study highlights that the secreted factors from M1 macrophages drive prostate cancer cell plasticity by upregulating the expression of CSC plasticity markers through NFκB signaling pathway.

In vivo vitamin D targets reveal the upregulation of focal adhesion-related genes in primary immune cells of healthy individuals.

Vitamin D modulates innate and adaptive immunity, the molecular mechanisms of which we aim to understand under human in vivo conditions. Therefore, we designed the study VitDHiD (NCT03537027) as a human investigation, in which 25 healthy individuals were supplemented with a single vitamin D3 bolus (80,000 IU). Transcriptome-wide differential gene expression analysis of peripheral blood mononuclear cells (PBMCs), which were isolated directly before and 24 h after supplementation, identified 452 genes significantly (FDR < 0.05) responding to vitamin D. In vitro studies using PBMCs from the same individuals confirmed 138 of these genes as targets of 1α,25-dihydroxyvitamin D3. A subset of the 91 most regulated in vivo vitamin D target genes indicated focal adhesion as the major pathway being upregulated by vitamin D3 supplementation of healthy individuals. Differences in the individual-specific responsiveness of in vivo vitamin D target genes in relation to the increase of the person's vitamin D status allowed a segregation of the VitDHiD participants into 9 high, 12 mid and 4 low responders. The expression profile of nearly 600 genes elucidate the difference between high and low vitamin D responders, the most prominent of which is the HLA-C (major histocompatibility complex, class I, C) gene.

Increased secretion of adipocyte-derived extracellular vesicles is associated with adipose tissue inflammation and the mobilization of excess lipid in human obesity.

BACKGROUND: Obesity is a worldwide epidemic characterized by adipose tissue (AT) inflammation. AT is also a source of extracellular vesicles (EVs) that have recently been implicated in disorders related to metabolic syndrome. However, our understanding of mechanistic aspect of obesity's impact on EV secretion from human AT remains limited. METHODS: We investigated EVs from human Simpson Golabi Behmel Syndrome (SGBS) adipocytes, and from AT as well as plasma of subjects undergoing bariatric surgery. SGBS cells were treated with TNFα, palmitic acid, and eicosapentaenoic acid. Various analyses, including nanoparticle tracking analysis, electron microscopy, high-resolution confocal microscopy, and gas chromatography-mass spectrometry, were utilized to study EVs. Plasma EVs were analyzed with imaging flow cytometry. RESULTS: EVs from mature SGBS cells differed significantly in size and quantity compared to preadipocytes, disagreeing with previous findings in mouse adipocytes and indicating that adipogenesis promotes EV secretion in human adipocytes. Inflammatory stimuli also induced EV secretion, and altered EV fatty acid (FA) profiles more than those of cells, suggesting the role of EVs as rapid responders to metabolic shifts. Visceral AT (VAT) exhibited higher EV secretion compared to subcutaneous AT (SAT), with VAT EV counts positively correlating with plasma triacylglycerol (TAG) levels. Notably, the plasma EVs of subjects with obesity contained a higher number of adiponectin-positive EVs than those of lean subjects, further demonstrating higher AT EV secretion in obesity. Moreover, plasma EV counts of people with obesity positively correlated with body mass index and TNF expression in SAT, connecting increased EV secretion with AT expansion and inflammation. Finally, EVs from SGBS adipocytes and AT contained TAGs, and EV secretion increased despite signs of less active lipolytic pathways, indicating that AT EVs could be involved in the mobilization of excess lipids into circulation. CONCLUSIONS: We are the first to provide detailed FA profiles of human AT EVs. We report that AT EV secretion increases in human obesity, implicating their role in TAG transport and association with adverse metabolic parameters, thereby emphasizing their role in metabolic disorders. These findings promote our understanding of the roles that EVs play in human AT biology and metabolic disorders.

DPYSL5 is highly expressed in treatment-induced neuroendocrine prostate cancer and promotes lineage plasticity via EZH2/PRC2.

Treatment-induced neuroendocrine prostate cancer (t-NEPC) is a lethal subtype of castration-resistant prostate cancer resistant to androgen receptor (AR) inhibitors. Our study unveils that AR suppresses the neuronal development protein dihydropyrimidinase-related protein 5 (DPYSL5), providing a mechanism for neuroendocrine transformation under androgen deprivation therapy. Our unique CRPC-NEPC cohort, comprising 135 patient tumor samples, including 55 t-NEPC patient samples, exhibits a high expression of DPYSL5 in t-NEPC patient tumors. DPYSL5 correlates with neuroendocrine-related markers and inversely with AR and PSA. DPYSL5 overexpression in prostate cancer cells induces a neuron-like phenotype, enhances invasion, proliferation, and upregulates stemness and neuroendocrine-related markers. Mechanistically, DPYSL5 promotes prostate cancer cell plasticity via EZH2-mediated PRC2 activation. Depletion of DPYSL5 decreases proliferation, induces G1 phase cell cycle arrest, reverses neuroendocrine phenotype, and upregulates luminal genes. In conclusion, DPYSL5 plays a critical role in regulating prostate cancer cell plasticity, and we propose the AR/DPYSL5/EZH2/PRC2 axis as a driver of t-NEPC progression.

Genetic variation is a key determinant of chromatin accessibility and drives differences in the regulatory landscape of C57BL/6J and 129S1/SvImJ mice.

Most common genetic variants associated with disease are located in non-coding regions of the genome. One mechanism by which they function is through altering transcription factor (TF) binding. In this study, we explore how genetic variation is connected to differences in the regulatory landscape of livers from C57BL/6J and 129S1/SvImJ mice fed either chow or a high-fat diet. To identify sites where regulatory variation affects TF binding and nearby gene expression, we employed an integrative analysis of H3K27ac ChIP-seq (active enhancers), ATAC-seq (chromatin accessibility) and RNA-seq (gene expression). We show that, across all these assays, the genetically driven (i.e. strain-specific) differences in the regulatory landscape are more pronounced than those modified by diet. Most notably, our analysis revealed that differentially accessible regions (DARs, N = 29635, FDR < 0.01 and fold change > 50%) are almost always strain-specific and enriched with genetic variation. Moreover, proximal DARs are highly correlated with differentially expressed genes. We also show that TF binding is affected by genetic variation, which we validate experimentally using ChIP-seq for TCF7L2 and CTCF. This study provides detailed insights into how non-coding genetic variation alters the gene regulatory landscape, and demonstrates how this can be used to study the regulatory variation influencing TF binding.

Seminal-Plasma-Mediated Effects on Sperm Performance in Humans.

Seminal plasma (SP) plays a crucial role in reproduction and contains a large number of proteins, many of which may potentially modify sperm functionality. To evaluate the effects of SP identity and its protein composition on human sperm function, we treated the sperm of several males with either their own or multiple foreign SPs in all possible sperm-SP combinations (full-factorial design). Then we recorded sperm motility and viability in these combinations and investigated whether the sperm performance is dependent on sperm and SP identity (or their interaction). Finally, we studied whether the above-mentioned sperm traits are affected by the abundance of three SP proteins, dipeptidyl peptidase-4 (DPP4), neutral endopeptidase (NEP), and aminopeptidase N (APN). The identity of the SP donor affected sperm swimming velocity, viability, and the proportion of hyperactivated sperm, but males' own SP was not consistently more beneficial for sperm than foreign SPs. Furthermore, we show that sperm performance is also partly affected by the interaction between sperm and SP donor. Finally, we found that DPP4 and NEP levels in SP were positively associated with sperm swimming velocity and hyperactivation. Taken together, our results highlight the importance of seminal plasma as a potential source of biomarkers for diagnostics and therapeutic interventions for male-derived infertility.

Fatty Acid Fingerprints and Hyaluronic Acid in Extracellular Vesicles from Proliferating Human Fibroblast-like Synoviocytes.

Extracellular vesicles (EVs) function as conveyors of fatty acids (FAs) and other bioactive lipids and can modulate the gene expression and behavior of target cells. EV lipid composition influences the fluidity and stability of EV membranes and reflects the availability of lipid mediator precursors. Fibroblast-like synoviocytes (FLSs) secrete EVs that transport hyaluronic acid (HA). FLSs play a central role in inflammation, pannus formation, and cartilage degradation in joint diseases, and EVs have recently emerged as potential mediators of these effects. The aim of the present study was to follow temporal changes in HA and EV secretion by normal FLSs, and to characterize the FA profiles of FLSs and EVs during proliferation. The methods used included nanoparticle tracking analysis, confocal laser scanning microscopy, sandwich-type enzyme-linked sorbent assay, quantitative PCR, and gas chromatography. The expression of hyaluronan synthases 1-3 in FLSs and HA concentrations in conditioned media decreased during cell proliferation. This was associated with elevated proportions of 20:4n-6 and total n-6 polyunsaturated FAs (PUFAs) in high-density cells, reductions in n-3/n-6 PUFA ratios, and up-regulation of cluster of differentiation 44, tumor necrosis factor α, peroxisome proliferator-activated receptor (PPAR)-α, and PPAR-γ. Compared to the parent FLSs, 16:0, 18:0, and 18:1n-9 were enriched in the EV fraction. EV counts decreased during cell growth, and 18:2n-6 in EVs correlated with the cell count. To conclude, FLS proliferation was featured by increased 20:4n-6 proportions and reduced n-3/n-6 PUFA ratios, and FAs with a low degree of unsaturation were selectively transferred from FLSs into EVs. These FA modifications have the potential to affect membrane fluidity, biosynthesis of lipid mediators, and inflammatory processes in joints, and could eventually provide tools for translational studies to counteract cartilage degradation in inflammatory joint diseases.

Sperm Physiological Response to Female Serum-Potential New Insights into the Reproductive Incompatibility Diagnostics.

Infertility is assumed to arise exclusively from male- and female-dependent pathological factors. However, recent studies have indicated that reproductive failure may also result from the reproductive incompatibility of the partners. Selection against such incompatibilities likely occurs via female-derived reproductive secretions, including follicular fluid (FF), that mediate gamete-level mate choice towards the sperm of specific males. To facilitate potential development of diagnostic tests for human reproductive incompatibility, we examined whether sperm physiological response to female serum indicate male-female compatibility in the presence of FF. We performed a full-factorial experiment, in which the sperm of 10 males were treated with the FF and serum of 6 healthy females. We found that sperm motility and viability in both biofluids were highly similar and that in 70% of the males, sperm serum treatment predicted male-female compatibility. We also identified male human leucocyte antigen (HLA) alleles and female (FF and serum) anti-HLA antibodies and tested whether the number of allele-antibody matches predict sperm physiological response to female fluids. However, no association was found between measured sperm traits and the number of allele-antibody matches. Overall, the present results may open novel possibilities for the future development of reproductive incompatibility tests and may pave the way towards more accurate infertility diagnostics and treatments.

Female-induced selective modification of sperm protein SUMOylation-potential mechanistic insights into the non-random fertilization in humans.

In many species, mate choice continues after the mating via female- or egg-derived biochemical factors that induce selective changes in sperm pre-fertilization physiology and behaviour. Recent studies have indicated that gamete-mediated mate choice likely occurs also in humans, but the mechanistic basis of the process has remained virtually unexplored. Here, we investigated whether female-induced modifications in sperm protein SUMOylation (post-translational modification of the proteome) could serve as a novel mechanism for gamete-mediated mate choice in humans. We treated the sperm of ten males with the oocyte-surrounding bioactive liquid (follicular fluid) of five females and investigated motility, viability and global protein SUMOylation status of the sperm in all (n = 50) of these male-female combinations (full-factorial design). All the measured sperm traits were affected by male-female combinations, and sperm protein SUMOylation status was also negatively associated with sperm motility. Furthermore, our results indicate that female-induced sperm protein SUMOylation is selective, potentially allowing females to increase sperm motility in some males, whereas decreasing it in the others. Consequently, our findings suggest that follicular fluid may non-randomly modify the structure and function of sperm proteome and in this way facilitate gamete-mediated mate choice in humans and possibly many other species. However, due to the relatively low number of female subjects and their potential infertility problems, our results should be replicated with larger subset of fully fertile women.

Chromatin-directed proteomics-identified network of endogenous androgen receptor in prostate cancer cells.

Treatment of prostate cancer confronts resistance to androgen receptor (AR)-targeted therapies. AR-associated coregulators and chromatin proteins hold a great potential for novel therapy targets. Here, we employed a powerful chromatin-directed proteomics approach termed ChIP-SICAP to uncover the composition of chromatin protein network, the chromatome, around endogenous AR in castration resistant prostate cancer (CRPC) cells. In addition to several expected AR coregulators, the chromatome contained many nuclear proteins not previously associated with the AR. In the context of androgen signaling in CRPC cells, we further investigated the role of a known AR-associated protein, a chromatin remodeler SMARCA4 and that of SIM2, a transcription factor without a previous association with AR. To understand their role in chromatin accessibility and AR target gene expression, we integrated data from ChIP-seq, RNA-seq, ATAC-seq and functional experiments. Despite the wide co-occurrence of SMARCA4 and AR on chromatin, depletion of SMARCA4 influenced chromatin accessibility and expression of a restricted set of AR target genes, especially those involved in cell morphogenetic changes in epithelial-mesenchymal transition. The depletion also inhibited the CRPC cell growth, validating SMARCA4's functional role in CRPC cells. Although silencing of SIM2 reduced chromatin accessibility similarly, it affected the expression of a much larger group of androgen-regulated genes, including those involved in cellular responses to external stimuli and steroid hormone stimulus. The silencing also reduced proliferation of CRPC cells and tumor size in chick embryo chorioallantoic membrane assay, further emphasizing the importance of SIM2 in CRPC cells and pointing to the functional relevance of this potential prostate cancer biomarker in CRPC cells. Overall, the chromatome of AR identified in this work is an important resource for the field focusing on this important drug target.

BCOR modulates transcriptional activity of a subset of glucocorticoid receptor target genes involved in cell growth and mobility.

Glucocorticoid (GC) receptor (GR) is a key transcription factor (TF) that regulates vital metabolic and anti-inflammatory processes. We have identified BCL6 corepressor (BCOR) as a dexamethasone-stimulated interaction partner of GR. BCOR is a component of non-canonical polycomb repressor complex 1.1 (ncPCR1.1) and linked to different developmental disorders and cancers, but the role of BCOR in GC signaling is poorly characterized. Here, using ChIP-seq we show that, GC induces genome-wide redistribution of BCOR chromatin binding towards GR-occupied enhancers in HEK293 cells. As assessed by RNA-seq, depletion of BCOR altered the expression of hundreds of GC-regulated genes, especially the ones linked to TNF signaling, GR signaling and cell migration pathways. Biotinylation-based proximity mapping revealed that GR and BCOR share several interacting partners, including nuclear receptor corepressor NCOR1. ChIP-seq showed that the NCOR1 co-occurs with both BCOR and GR on a subset of enhancers upon GC treatment. Simultaneous depletion of BCOR and NCOR1 influenced GR target gene expression in a combinatorial and gene-specific manner. Finally, we show using live cell imaging that the depletion of BCOR together with NCOR1 markedly enhances cell migration. Collectively, our data suggest BCOR as an important gene and pathway selective coregulator of GR transcriptional activity.

SUMOylation regulates the protein network and chromatin accessibility at glucocorticoid receptor-binding sites.

Glucocorticoid receptor (GR) is an essential transcription factor (TF), controlling metabolism, development and immune responses. SUMOylation regulates chromatin occupancy and target gene expression of GR in a locus-selective manner, but the mechanism of regulation has remained elusive. Here, we identify the protein network around chromatin-bound GR by using selective isolation of chromatin-associated proteins and show that the network is affected by receptor SUMOylation, with several nuclear receptor coregulators and chromatin modifiers preferring interaction with SUMOylation-deficient GR and proteins implicated in transcriptional repression preferring interaction with SUMOylation-competent GR. This difference is reflected in our chromatin binding, chromatin accessibility and gene expression data, showing that the SUMOylation-deficient GR is more potent in binding and opening chromatin at glucocorticoid-regulated enhancers and inducing expression of target loci. Blockage of SUMOylation by a SUMO-activating enzyme inhibitor (ML-792) phenocopied to a large extent the consequences of GR SUMOylation deficiency on chromatin binding and target gene expression. Our results thus show that SUMOylation modulates the specificity of GR by regulating its chromatin protein network and accessibility at GR-bound enhancers. We speculate that many other SUMOylated TFs utilize a similar regulatory mechanism.

Common and personal target genes of the micronutrient vitamin D in primary immune cells from human peripheral blood.

Vitamin D is essential for the function of the immune system. In this study, we treated peripheral blood mononuclear cells (PBMCs) of healthy adults with the biologically active form of vitamin D3, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) using two different approaches: single repeats with PBMCs obtained from a cohort of 12 individuals and personalized analysis based on triplicates of five study participants. This identified 877 (cohort approach) and 3951 (personalized approach) genes that significantly (p < 0.05) changed their expression 24 h after 1,25(OH)2D3 stimulation. From these, 333 and 1232 were classified as supertargets, a third of which were identified as novel. Individuals differed largely in their vitamin D response not only by the magnitude of expression change but also by their personal selection of (super)target genes. Functional analysis of the target genes suggested the overarching role of vitamin D in the regulation of metabolism, proliferation and differentiation, but in particular in the control of functions mediated by the innate and adaptive immune system, such as responses to infectious diseases and chronic inflammatory disorders. In conclusion, immune cells are an important target of vitamin D and common genes may serve as biomarkers for personal responses to the micronutrient.

BCOR-coupled H2A monoubiquitination represses a subset of androgen receptor target genes regulating prostate cancer proliferation.

We have identified BCL6 corepressor (BCOR) as a hormone-dependent interaction partner of androgen receptor (AR), a key transcription factor in the development of normal and cancerous prostate. BCOR is often mutated in cancers and hematological diseases and as a component of a non-canonical polycomb repressive complex 1 (ncPRC1.1) required for arranging many facets of cellular differentiation. However, its role in androgen signaling or prostate cancer cells remains unknown. Here, our genome-wide analyses reveal that BCOR is recruited in an androgen-dependent fashion to majority of AR-binding chromatin sites in castration-resistant prostate cancer (CRPC) cells. Interestingly, depletion of BCOR has a significant effect on the expression of androgen-repressed genes linked to regulation of cell proliferation, differentiation and development. At many of these genes, such as HOX genes, the depletion leads to a decrease in H2A K119 monoubiquitination and an increase in mRNA expression. Consistently, BCOR depletion impairs the proliferation and viability of CRPC cells, inducing their apoptosis. Collectively, our data indicate a key role for the BCOR-ncPRC1.1 complex in the corepression of an important subset of AR target genes and the regulation of prostate cancer cell proliferation.

A long hypoxia-inducible factor 3 isoform 2 is a transcription activator that regulates erythropoietin.

Hypoxia-inducible factor (HIF), an αß dimer, is the master regulator of oxygen homeostasis with hundreds of hypoxia-inducible target genes. Three HIF isoforms differing in the oxygen-sensitive α subunit exist in vertebrates. While HIF-1 and HIF-2 are known transcription activators, HIF-3 has been considered a negative regulator of the hypoxia response pathway. However, the human HIF3A mRNA is subject to complex alternative splicing. It was recently shown that the long HIF-3α variants can form αß dimers that possess transactivation capacity. Here, we show that overexpression of the long HIF-3α2 variant induces the expression of a subset of genes, including the erythropoietin (EPO) gene, while simultaneous downregulation of all HIF-3α variants by siRNA targeting a shared HIF3A region leads to downregulation of EPO and additional genes. EPO mRNA and protein levels correlated with HIF3A silencing and HIF-3α2 overexpression. Chromatin immunoprecipitation analyses showed that HIF-3α2 binding associated with canonical hypoxia response elements in the promoter regions of EPO. Luciferase reporter assays showed that the identified HIF-3α2 chromatin-binding regions were sufficient to promote transcription by all three HIF-α isoforms. Based on these data, HIF-3α2 is a transcription activator that directly regulates EPO expression.

IRF2BP2 modulates the crosstalk between glucocorticoid and TNF signaling.

IRF2BP2 (interferon regulatory factor-2 binding protein-2) is an uncharacterized interaction partner of glucocorticoid (GC) receptor (GR), an anti-inflammatory and metabolic transcription factor. Here, we show that GC changes the chromatin binding of IRF2BP2 in natural chromatin milieu. The GC-induced IRF2BP2-binding sites co-occur with GR binding sites and are associated with GC-induced genes. Moreover, the depletion of IRF2BP2 modulates transcription of GC-regulated genes, represses cell proliferation and increases cell movement in HEK293 cells. In A549 cells, the depletion extensively alters the responses to GC and tumor necrosis factor α (TNF), including metabolic and inflammatory pathways. Taken together, our data support the role of IRF2BP2 as a coregulator of both GR and NF-κB, potentially modulating the crosstalk between GC and TNF signaling.

Lack of androgen receptor SUMOylation results in male infertility due to epididymal dysfunction.

Androgen receptor (AR) is regulated by SUMOylation at its transactivation domain. In vitro, the SUMOylation is linked to transcriptional repression and/or target gene-selective regulation. Here, we generated a mouse model (ArKI) in which the conserved SUMO acceptor lysines of AR are permanently abolished (ArK381R, K500R). ArKI males develop normally, without apparent defects in their systemic androgen action in reproductive tissues. However, the ArKI males are infertile. Their spermatogenesis appears unaffected, but their epididymal sperm maturation is defective, shown by severely compromised motility and fertilization capacity of the sperm. Fittingly, their epididymal AR chromatin-binding and gene expression associated with sperm maturation and function are misregulated. AR is SUMOylated in the wild-type epididymis but not in the testis, which could explain the tissue-specific response to the lack of AR SUMOylation. Our studies thus indicate that epididymal AR SUMOylation is essential for the post-testicular sperm maturation and normal reproductive capability of male mice.

In vivo transcriptome changes of human white blood cells in response to vitamin D.

In the vitamin D intervention study VitDbol (NCT02063334) blood samples were drawn directly before an oral bolus (2000 µg vitamin D3) and 24 h later. The focus of phase II of VitDbol was the transcriptome-wide analysis of the effects of vitamin D gene expression in human peripheral blood mononuclear cells (PBMCs). All five participants responded in an individual fashion to the bolus by increases in serum levels of the vitamin D metabolites 25-hydroxyvitamin D3 (25(OH)D3) and 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). RNA sequencing identified 15.040 commonly expressed genes in PBMCs, 702 (4,7%) of which were significantly (p < 0,05) affected by the vitamin D3 bolus. KEGG pathway analysis suggested that these genes are involved in general protein translation, monocyte differentiation and cellular growth control. Previously published transcriptome-wide studies in comparable cell systems confirmed 234 of the 702 vitamin D target genes, leaving many genes, such as HLA-A and HLA-C, as novel discoveries. Interestingly, in vivo stimulated PBMCs of this study showed a larger number of common vitamin D target genes with the monocytic cell line THP-1 than with in vitro stimulated PBMCs. The expression pattern of vitamin D target genes differed significantly between individuals and the average expression change can serve as a marker for vitamin D responsiveness. In conclusion, this study demonstrates that under in vivo conditions changes in 25(OH)D3 and 1,25(OH)2D3 serum concentrations alter the expression of more than 700 vitamin D target genes in human leukocytes.

Crosstalk between androgen and pro-inflammatory signaling remodels androgen receptor and NF-κB cistrome to reprogram the prostate cancer cell transcriptome.

Inflammatory processes and androgen signaling are critical for the growth of prostate cancer (PC), the most common cancer among males in Western countries. To understand the importance of potential interplay between pro-inflammatory and androgen signaling for gene regulation, we have interrogated the crosstalk between androgen receptor (AR) and NF-κB, a key transcriptional mediator of inflammatory responses, by utilizing genome-wide chromatin immunoprecipitation sequencing and global run-on sequencing in PC cells. Co-stimulation of LNCaP cells with androgen and pro-inflammatory cytokine TNFα invoked a transcriptome which was very distinct from that induced by either stimulation alone. The altered transcriptome that included gene programs linked to cell migration and invasiveness was orchestrated by significant remodeling of NF-κB and AR cistrome and enhancer landscape. Although androgen multiplied the NF-κB cistrome and TNFα restrained the AR cistrome, there was no general reciprocal tethering of the AR to the NF-κB on chromatin. Instead, redistribution of FOXA1, PIAS1 and PIAS2 contributed to the exposure of latent NF-κB chromatin-binding sites and masking of AR chromatin-binding sites. Taken together, concomitant androgen and pro-inflammatory signaling significantly remodels especially the NF-κB cistrome, reprogramming the PC cell transcriptome in fashion that may contribute to the progression of PC.

Global analysis of transcription in castration-resistant prostate cancer cells uncovers active enhancers and direct androgen receptor targets.

Androgen receptor (AR) is a male sex steroid-activated transcription factor (TF) that plays a critical role in prostate cancers, including castration-resistant prostate cancers (CRPC) that typically express amplified levels of the AR. CRPC-derived VCaP cells display an excessive number of chromatin AR-binding sites (ARBs) most of which localize to distal inter- or intragenic regions. Here, we analyzed direct transcription programs of the AR in VCaP cells using global nuclear run-on sequencing (GRO-seq) and integrated the GRO-seq data with the ARB and VCaP cell-specific TF-binding data. Androgen immediately activated transcription of hundreds of protein-coding genes, including IGF-1 receptor and EGF receptor. Androgen also simultaneously repressed transcription of a large number of genes, including MYC. As functional enhancers have been postulated to produce enhancer-templated non-coding RNAs (eRNAs), we also analyzed the eRNAs, which revealed that only a fraction of the ARBs reside at functional enhancers. Activation of these enhancers was most pronounced at the sites that also bound PIAS1, ERG and HDAC3, whereas binding of HDAC3 and PIAS1 decreased at androgen-repressed enhancers. In summary, our genome-wide data of androgen-regulated enhancers and primary target genes provide new insights how the AR can directly regulate cellular growth and control signaling pathways in CPRC cells.